Bottom Line:
Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104.Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport.Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

Affiliation: Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, Missouri, United States of America.

ABSTRACTAxonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

Mentions:
To address how the unc-104(gf) suppresses the SV trafficking defects of sam-4, we characterized the two unc-104 alleles in the absence of sam-4. In isolation, js1288 and js1289 show grossly normal mechanosensory neuron anatomy (Figure 7F, 7G). We analyzed their effects on transport by examining GFP-RAB-3 distribution in vivo. We found that GFP-RAB-3 accumulations are significantly increased in the distal part of PLM neurites (Figure 7E–7H) in each of these unc-104 mutants but decreased in the soma (Figure 7E″–7G″, 7J), indicating that SV transport is enhanced by these two mutations. However, we did not observe GFP-RAB-3 increase in PLM synaptic varicosities (Figure 7E′–7G′, 7I). This is probably because either SV levels in PLM varicosities are already saturated in the wild type background or other mechanisms exist at pre-synapses to maintain SV homeostasis. To further understand how these mutations affect SV dynamics, we examined GFP-RAB-3 trafficking using live imaging (Figure 8). We found that both mutations result in increased run length of GFP-RAB-3 transport (Figure 8E). We also noticed that js1289 results in greater flux of GFP-RAB-3 (Figure 8F), while jsIs1288 reduces SV transport velocity (Figure 8G). Thus, processivity of vesicle transport is increased in both gain-of-function mutants, though perhaps by distinct mechanisms. Western blot analysis of protein levels showed that neither of these two lesions alter UNC-104 protein levels in vivo (Figure S9B). Hence, increasing processivity of the SV transport through the UNC-104 motor domain can partially bypass the need for SAM-4. This is consistent with our hypothesis that SAM-4 functions through the UNC-104 motor domain to regulate SV transport.

Mentions:
To address how the unc-104(gf) suppresses the SV trafficking defects of sam-4, we characterized the two unc-104 alleles in the absence of sam-4. In isolation, js1288 and js1289 show grossly normal mechanosensory neuron anatomy (Figure 7F, 7G). We analyzed their effects on transport by examining GFP-RAB-3 distribution in vivo. We found that GFP-RAB-3 accumulations are significantly increased in the distal part of PLM neurites (Figure 7E–7H) in each of these unc-104 mutants but decreased in the soma (Figure 7E″–7G″, 7J), indicating that SV transport is enhanced by these two mutations. However, we did not observe GFP-RAB-3 increase in PLM synaptic varicosities (Figure 7E′–7G′, 7I). This is probably because either SV levels in PLM varicosities are already saturated in the wild type background or other mechanisms exist at pre-synapses to maintain SV homeostasis. To further understand how these mutations affect SV dynamics, we examined GFP-RAB-3 trafficking using live imaging (Figure 8). We found that both mutations result in increased run length of GFP-RAB-3 transport (Figure 8E). We also noticed that js1289 results in greater flux of GFP-RAB-3 (Figure 8F), while jsIs1288 reduces SV transport velocity (Figure 8G). Thus, processivity of vesicle transport is increased in both gain-of-function mutants, though perhaps by distinct mechanisms. Western blot analysis of protein levels showed that neither of these two lesions alter UNC-104 protein levels in vivo (Figure S9B). Hence, increasing processivity of the SV transport through the UNC-104 motor domain can partially bypass the need for SAM-4. This is consistent with our hypothesis that SAM-4 functions through the UNC-104 motor domain to regulate SV transport.

Bottom Line:
Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104.Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport.Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.

Affiliation:
Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, Missouri, United States of America.

ABSTRACTAxonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.